The determination of the presence of polymorphisms, especially mutations, in DNA has become a very important and useful tool for a variety of purposes. Detecting mutations which are known to cause or to predispose persons to disease is one of the more important uses of determining the possible presence of a mutation. One example is the analysis of the gene named BRCA1 which may result in breast cancer if it is mutated (Miki et al., 1994; Futreal et al., 1994). Several known mutations in the BRCA1 gene have been causally linked with breast cancer. With this knowledge in hand it is now possible to screen women for these known mutations to determine whether they are predisposed to develop breast cancer. Some other uses for determining polymorphisms or mutations are for genotyping and for mutational analysis for positional cloning experiments.
A few different methods are commonly used to analyze DNA for polymorphisms or mutations. The most definitive method is to sequence the DNA to determine the actual base sequence (Maxam and Gilbert, 1977; Sanger et al., 1977). Although such a method is the most definitive it is also the most expensive and time consuming method. Restriction mapping analysis has some limited use in analyzing DNA for polymorphisms. If one is looking for a known polymorphism at a site which will change the recognition site for a restriction enzyme it is possible simply to digest DNA with this restriction enzyme and analyze the fragments on a gel or with a Southern blot to determine the presence or absence of the polymorphism. This type of analysis is also useful for determining the presence or absence of gross insertions or deletions. Hybridization with allele specific oligonucleotides is yet another method for determining the presence of known polymorphisms. These latter methods require the use of hybridization techniques which are time consuming and costly.
In recent years some breakthroughs have been made which allow the use of mass spectrometry to analyze macromolecules (Hillenkamp et al., 1991; Schneider and Chait, 1995; Wang and Biemann, 1994; Nordhoffet al., 1993; Siuzdak, 1994; Wu et al., 1994; Nelson et al., 1989; Nordhoff et al., 1994; Kirpekar et al., 1994; and Pieles et al., 1993). Many papers have now been published which establish that mass spectrometry can be used to analyze DNA fragments. Some papers have focused simply on the ability to measure the mass of a single fragment of DNA or RNA whereas others have gone so far as to show the utility of mass spectrometry for sequencing short fragments of nucleic acids (Pieles et al., 1993; Kirpekar et al., 1994). In all of the papers published to date the analysis has been limited to polynucleotides on the order of about 50 nucleotides or fewer. The appeal of mass spectrometry is the tremendous speed in obtaining data. Once samples have been prepared the throughput can be as fast as 1-2 seconds per sample. Analysis of the data is then done off-line. This time of 1-2 seconds is a tremendous advantage when compared with the many hours needed for running gels and/or hybridizing samples for analysis if the more classical methods of nucleic acid polymorphism analysis are used.
The present invention applies mass spectrometry to the determination of the presence of polymorphisms within known genes. The method uses mass spectrometry to compare the mass spectrum of a fragment of DNA from a sample to be analyzed with known reference mass spectra of DNA, e.g., spectra for wild-type DNA and DNA with a known polymorphism. Determination of the presence of a polymorphism in the sample being tested is rapid and accurate. The use of mass spectrometry with its very rapid analysis is especially useful for routine screening of large numbers of samples.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the text and respectively grouped in the appended List of References.